CHARACTERIZATION OF STRUCTURE AND FUNCTION OF HISTONES IN EMBRYONIC STEM CELLS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 已经提出，染色质参与的表观遗传机制影响效应蛋白的可及性，并且通过由组蛋白的组合共价修饰引起的染色质结构的修饰来调节重要的下游细胞活性。对哺乳动物组蛋白的修饰及其功能的全面表征仍然是一个挑战，因为它的复杂性，特别是对它们的修饰的压倒性数量。本项目首先采用色谱法分离组蛋白，然后（1）采用自顶向下的FTICR/ECD/MS策略直接表征H1、H2、H4上的修饰，（2）采用我们的筛选策略和自中而下的FTICR/ECD/MS技术综合表征修饰，我们将通过基因缺失和RNA干扰技术扰乱生物系统，然后采用我们的质谱分析策略来表征由于基因缺失和RNA干扰而引起的所有修饰变化。质谱法将在这个项目中发挥核心和不可或缺的作用。 没有其他替代方法，我们可以全面表征所有的修饰，以阐明其生物学功能。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. It has been suggested that chromatin-involved epigenetic mechanisms affect the accessibility of effector proteins and regulate important downstream cellular activities via modifications of chromatin structure resulting from combinatorial covalent modifications of histones.Despite its significance, comprehensive characterization of modifications on mammalian histones and their function remains a challenge because of its complexity especially the overwhelming number of modifications on them.In this project, firstly, we will separate the histones by chromatography first and then (1) apply topdown FTICR/ECD/MS strategy to directly characterize modifications on H1, H2, H4 and (2) apply our screening strategy and middle-down FTICR/ECD/MS technique to comprehensively characterize the modifications, we will perturb the biological system by gene deletion and RNA interference techniques and then employ our mass spectrometry strategies to characterize all the induced changes in modification due to the perturbation.Mass spectrometry will play a central and indispensable role in this project. There are no other alternative methods by which we can comprehensively characterize all the modifications in order to elucidate their biological functions.